The kallikreins-kinins system is composed of two major proteolytic systems (in plasma and tissues) that are responsible for the liberation of pro-inflammatory substances, bradykinin (BK) and kallidin (LysBK). Kinins are formed from precursors (high and low molecular weight kininogens, HMWK and LMWK) by the action of serine-proteases named kallikreins. Two types of kallikreins may generate kinins, the first, from hepatic origin, is present in circulating blood and the other is found in many tissues and exocrine glands. Main substrate for plasma kallikrein is the HMWK, the precursor of the nonapeptide BK. Tissue kallikreins originate from many cell types (epithelial and endothelial cells, smooth muscles, blood cells, neurones, etc) and belong to a polygenic family. In vitro, tissue prekallikreins may be converted into active kallikreins by some proteases such as trypsin and plasmin. Tissue kallikreins preferentially cleave the LMWK to generate LysBK. LysBK can be converted to BK by aminopeptidases. BK and LysBK may be further converted by carboxypeptidases of the M type (CPM) and the N type (CPN) into metabolites devoid of the C-terminal arginyl residue (desArg9BK and Lys-desArg9BK) which display distinct biological activities mediated by a receptor type called BKB1 (see below). It has also been shown that activated mast cells, basophils or neutrophils secrete substances with kallikrein-like activities; for instance elastases that enable the formation of kinins (Bhoola et al. 1992, Pharmacol Rev 44, 1–80). Half life of kinins is estimated to be less than 30 sec in human. The short lasting effects of kinins in vivo have been attributed to the rapid degradation of these peptides by proteolytic enzymes circulating in blood or anchored to cell surfaces, especially of the endothelium. Peptidases involved in kinin inactivation are called kininases. The importance of each enzyme in kinin metabolism differ from species to species, from tissue to tissue, and also depends on the conditions (normal vs. inflammed) prevailing in tissues under physiological or pathological states. The most active enzymes that are believed to be involved in kinin degradation are enzyme commission numbers [3.4.11.2]; [3.4.11.9]; [3.4.13.9]; [3.4.13.8]; [3.4.24.11]; [3.4.15.1]; [3.4.24.15]; [3.4.24.16]; [3.4.21.1]; [3.4.17.1]; and [3.4.21.26].
Kinins can be degraded by three categories of enzymes namely the aminopeptidases, the endopeptidases and the carboxypeptidases. Hydrolytic actions of endopeptidases yield inactive fragments. The action of aminopeptidase M (AmM) does not influence the biological activities of bradykinin B2 (BKB2) receptor agonists since BK and LysBK are equally active; however, deletion of N-terminal Lys causes a drastic loss of activity on the human BKB1 receptor because Lys-desArg9BK is 100 fold more potent than desArg9BK (Gobeil et al. 1996, Br J Pharmacol 118, 289–294). Among kininases, the angiotensin converting enzyme (ACE, alias kininase II) and the CPN (Kininase I) have been extensively studied. Both enzymes are zinc-metalloproteases; the former being strongly expressed in lung endothelial cells (and many other tissues; smooth muscles, epithelia and fibroblasts), the latter being confined to the bloodstream. While ACE is responsible for the production of inactive kinin fragments, the plasma CPN and tissue CPM are implicated in the release of active metabolites acting at the BKB1 receptor. Interestingly one of this ectoenzymes, the CPM, can be up regulated (in some experimental pathologies such as endotoxemia), a biological phenomenon that parallels the induction of BKB1 receptors as well as the enhanced formation of BKB1 receptor stimulants in the cardiovascular system and tissue compartments (Erdös and Skidgel, 1997, The kinin system, Academic Press, London UK, pp 111–141).
Receptors for Kinins
The various actions of BK, LysBK and their metabolites are mediated by two receptor subtypes, called BKB1 and BKB2 (Regoli and Barabé, 1980, Pharmacol Rev 32, 1–46). These are rhodopsin-like proteins which coupled to various G proteins that differ from one to another target cell. BKB2 receptor is constitutive and is found in a variety of cells as endothelia, smooth muscles, epithelia, white blood cells: it mediates smooth muscle contraction and the release of autacoids, particularly from the endothelium: this function provides the basic mechanism of peripheral vasodilatation which is responsible for a large part of the in vivo hypotensive effect. Similar to BKB1, the BKB2 receptor has been cloned in human, rabbit, rat and mouse: the BKB2 are small proteins composed of 360–367 amino acids, slightly larger than the BKB1 receptor, because of the presence of a larger C-terminal cytoplasmic segment. The BKB2 receptor has been characterized by mean of selective agonists and antagonists in pharmacological and biochemical assays, using native and recombinant receptors from various species (Regoli et al. 1998, Eur J Pharm 348, 1–10).
The BKB1 receptor is usually not found in physiological conditions but it is induced by various stimuli in several cell types including endothelial, smooth muscle and blood cells, neurons (Marceau et al. 1998, Pharmacol Rev 50, 357–386). BKB1 receptor activation is not as rapid as that of the BKB2 and the effects are more stable and slowly reversible than those of the BKB2 receptor. According to Marceau et al (supra), “the specific role of BKB1 receptor induction may be to amplify the tissue effects of kinins as a function of time via the synthesis of novel receptor molecules that are resistant to tachyphylaxis”. BKB1 receptors of human, rabbit, rat and mouse have been cloned and shown to be small proteins of 334–353 amino acids. They mediate contraction of vascular and other smooth muscles as well as the decrease of blood pressure, which in large part is due to peripheral vasodilatation when desArg9BK is applied intravenously in LPS-treated rabbits. BKB1 receptors have been characterized with selective agonists and antagonists, using the classical criteria of the order of potency of agonists and the values of apparent affinities of competitive antagonists.
The Kallikrein-Kinin System in Pathophysiology
The kallikrein-kinin system has been implicated in a variety of physiological functions as well as of pathological states. Indeed, the involvement of kinins has been reported both in the acute and in the chronic phases of inflammation, in pain, in septic inflammatory response syndrome (SIRS), in pancreatitis, in asthma and allergic rhinitis; moreover a protective role of kinins in myocardial ischemia has been suggested (Farmer et al., 1997, The Kinin System, Academic Press, London UK).
In inflammation and pain, tumor, rubor, calor and lesa function are the four cardinal signs of inflammation and exogenous kinin BKB1 and BKB2 receptor agonists can produce all of them when applied to animal and human tissues. Tumor, rubor, calor derive from arterial vasodilatation and the endothelium contraction at the postcapillary veins, while pain is due to activation of receptors in the sensory nerves and the potentiating effect of prostaglandins. Kinin contribute to the acute tissue reaction to noxious stimuli by promoting the release of histamine and, in some species, serotonin from mast cells and that of neuropeptides (e.g. substance P) from sensory nerves: they also contribute to tissue repair by activating cell division, motility and functions of tissue and blood components as macrophages, fibroblasts (e.g. from foetal human lung) and the synthesis of collagen. BKB2 receptors appear to be implicated in the early stage and BKB1 receptors in the late stage of the inflammatory process. Inducible BKB1 receptors are involved in various types of inflammation, diabetic and other types of vasculopathies, pain and angiogenesis (Marceau et al. supra). Recent reports point to an important role of kinin BKB1 receptors in physiopathology. Dray and Perkins (1993, Trends Neurosci 16, 99–104) have reviewed the possible implication of BKB1 receptors in various inflammatory states, tissue reactions to noxious stimuli and hyperalgesia, particularly the chronic phases of these disturbancies. This has been further supported by recent findings from Davis and coworkers (Davis et al. 1994, J Med Biol Res 27, 1793–1802) in inflammatory hyperalgesia in the rat. As pointed out by Marceau (1995, Immunopharmacol 30, 1–26), “it is conceivable that BKB1 receptors can amplify the responses of injured tissues to kinins and, in some cases, take the relay of BKB2 receptors in chronic pathologies”.
Peptidic BKB1 receptor antagonists have been developed in the late seventies (Regoli et al. 1977, Can J Physiol Pharmacol 55, 855–867; Regoli and Barabé´ 1980, J Pharmacol Rev 32, 1–46). Some progress has been made and peptidic compounds have been improved (Gobeil et al., 1996, Hypertension 28, 833–839; Gobeil et al. 1999, Hypertension 33, 823–829; Neugebauer et al. 2002, Can J Physiol Pharmacol 80, 287–292). Other compounds have been discovered, of peptidic and non-peptidic nature. BKB1 receptor antagonists have been disclosed in various published patent documents (WO97/09346, U.S. Pat. Nos. 6,075,120, US5,863,899, US5,849,863, US5,843,900, US5,834,431, US5,750,506, US5,700,779, US5,635,593, US5,610,140, WO97/25315, WO98/07746, WO00/75107, WO01/05783, WO02/, U.S. Pat. Nos. 6,468,972, US6,241,993, US5,849,312) as well as in non-patent documents (Stewart et al., 1997, Can J Physiol Pharmacol 75: 719–724; Larrivée et al., 2000, Brit J Pharmacol 131: 885–892; Mason et al., 2002, Can J Physiol Pharmacol 80: 264–268; Miskolzie et al., 2002, J Biomol Struct Dyn 19: 585–593; Horlick et al., 1999, Immunopharmacol 43: 169–177; Neugebauer et al, 2002, supra; Bedos et al., 2000, J Med Chem 43: 2387–2394)
Despite the antagonists of the prior art, it would still be highly desirable to provide BKB1 receptor antagonists, that are potent, selective and specific for the BKB1 receptor, and resistant to proteolytic degradation with chemical features that favour their absorption and distribution in the body in order to improve potency and duration of action in vivo.